There is an increasing demand for a simple but accurate and reproducible method for determining relatively low concentrations e.g., 0.025, 0.05, 0.10 or 0.20% of ethanol in aqueous fluids. One common application of such a method could be to measure ethanol levels in human body fluids as a test for highway intoxication or sobriety. Another would be to assure that operators of dangerous equipment, such as heavy construction equipment or military equipment are not intoxicated by alcohol. The chemical methods of choice for such tests include complex laboratory procedures such as gas chromatography for analyzing blood or urine, and a range of laboratory or field colorimetric tests. Since the late 1930's the "Drunkometer" of Stephenson Corporation has been in use. This colorimetric machine relies on the ability of ethanol in the breath to react with and discolor aqueous acidic potassium permanganate. In 1954 the Stephenson Corporation introduced the widely used "Breathalyzer" which relies on the decolorizing reaction of ethanol in the breath with acidic potassium dichromate to determine intoxication. These tests have a long history of use which has not been without difficulty and controversy. They require difficult to carry wet reagents, calibration and a reasonable level of operator competence to give good readings They are not readily suited for rapid routine processing of large groups of samples.
Another application for the rapid determination of blood alcohol levels is in the hospital emergency rooms. Approximately 1/3 of all patients currently admitted to hospital emergency rooms are tested for blood alcohol level for the purpose of making a correct judgment as to the nature of their clinical condition. Since no simple rapid method has previously existed for such a measurement current practice is to take a blood sample by venipuncture and to hand carry it to the laboratory for a stat blood alcohol determination. This procedure takes from 30 minutes to a few hours.
The present invention employs the enzyme alcohol oxidase. This material was reported by Janssen et al in Biochem Biophys Res Commun (US) 8 Sept. 1965, 20, (5) p 630-4 and is presently sold by Boehringer Mannheim Biochemicals, Indianapolis, IN and Phillips Chemical, a subsidiary of Phillips Petroleum.
Alcohol oxidase is a particularly unstable enzyme undergoing rapid deterioration and loss of activity. It is sold by Phillips as a solution in 70% by weight sucrose. It is sold by Boehringer as a solid, stabilized with large amounts of the peroxide antagonist, reduced glutathione. This is not an acceptable form of this analysis which requires that hydrogen peroxide be generated and quantitated. The large amounts of peroxide antagonist would interfere with this. In addition, it has also been found that this enzyme has the interesting but difficult to deal with property of undergoing autoxidation, generating peroxide by reaction with itself.
This enzyme's use in electrode ethanol analysis systems has been reported in Chem Abstracts, 94, 135580a, and is mentioned in U.S. Pat. No. 4,250,261 of Eggeling et al. Majkic-Singh and Berkes reported at Analytica Chemica Acta, 115, 401-5 (1980) a method for determining ethanol in which the chromogen 2,2'-azino-di(3-ethylbenzthiazoline-6-sulfonate) (ABTS) was reacted with hydrogen peroxide generated by alcohol oxidase. This prior method is carried out measuring the absorbency of solutions in a spectrophotometer in a laboratory setting. It is also characterized by careful handling of the sensitive alcohol oxidase in an effort to prevent deterioration or variation of results.
U.S. Pat. No. 4,430,427 of Hopkins also employs ethanol oxidase in a solution analysis setting.
What is now sought and provided by the present invention is a long-lived accurate and precise simple colorimetric method for determining ethanol concentrations in aqueous fluids such as human body fluids, in particular saliva and whole blood.